Project Summary In essentially all lineages, the cell cycle is quiescent in stem cells, and undergoes mitotic exit in terminally differentiated cells. In the intervening developmental period, however, cell cycles have been regarded as ?generic?, regulated only with respect to their number, so as to maintain homeostasis or respond to stress. The generic view of the mammalian cell cycle contrasts with the specialized cell cycles of early embryonic development in model organisms such as Drosophila or Xenopus, where cell cycle control, including dramatic changes in cell cycle length, are intimately linked to developmental events. Our recently published work, including a single-cell transcriptomic analysis of the mouse erythroid trajectory and a study of replication fork dynamics in early erythropoiesis has uncovered the presence of specialized cell cycles throughout mammalian erythroid development. Our principal hypothesis is that developmental-stage-specific specializations of the cell cycle are integral to the process of differentiation, and regulate both incremental changes such as cell growth, as well as switch-like cell fate decisions. In this proposal, we investigate cell cycle specialization in early erythropoiesis, orchestrated around the time of a key cell fate switch, from self-renewal of CFU-e progenitors, to erythroid terminal differentiation (ETD). We found that, preceding this switch, there is progressive shortening of G1; and that, at the switch, there is an abrupt shortening of S phase. Further, S phase shortening is the result of a novel mechanism of regulating S phase length, through a global increase in replication fork speed. In this proposal, we will investigate both the mechanisms, as well as the functional outcomes, of these cell cycle specializations. In AIM 1, we will carry out functional analysis of four erythroid regulators: E2F4, KLF1, EpoR and Stat5. Using mice mutant for each of these regulators, we will determine their roles in erythroid cell cycle specializations and consequent developmental decisions. In AIM 2, we will carry out single-cell RNA-seq analysis of progenitors deleted for each of the four regulators. We will order cell transcriptomes to generate the erythroid developmental pesudotime, and determine abnormalities along this pseudotime, including failure to upregulate replication genes, abnormal cell densities that might reflect developmental delays or arrest, and cell cycle phase for each cell. We will correlate any abnormalities at the single cell level. In AIM 3, we will determine whether S phase shortening is required for the CFU-e / ETD switch, using a variety of drugs and genetic manipulation to prevent, or accelerate, S phase shortening, and examine the consequent effect on the CFU-e/ETD switch. Further, we will examine the potential role of S phase shortening in modifying chromatin accessibility at the CFU- e/ETD switch. IMPACT: this proposal deals with innovative cell cycle modifications that might directly regulate the developmental process. Specifically, delaying the CFU-e/ETD switch with cell cycle modifying drugs results in amplification of CFU-e, a translational goal in the treatment of anemia.